Clostridium botulinum produces botulinum neurotoxin (BoNT), the causative agent of the debilitating disease botulism that affects humans and animals. BoNTs have also been considered as potential bioterrorism (BT) agents. For instance, in 2001 C. botulinum was listed as a category A select agent due to its potential use as a BT agent [1]. In addition to its importance as a pathogen and potential BT agent, BoNTs are now widely used as pharmaceuticals to treat a myriad of neuronal disorders.
Seven distinguishable serotypes of BoNT, designated A-G, are produced by C. botulinum. The serotypes are described as BoNT/A, BoNT/B, BoNT/C, etc. BoNTs are produced as progenitor toxin complexes in which the neurotoxin is associated with nontoxic components: nonhemagglutinin (NTNH), hemagglutinin (HA), other uncharacterized protein components, and nucleic acids [2]. The genes encoding the neurotoxin and associated protein components of the toxin complexes are organized in a cluster, and their location and composition varies among the different serotypes and strains [3]. For further information concerning the properties of the various C. botulinum toxins, reference is made to the article by Jankovic and Brin, The New England Journal of Medicine, No. 17, 1990, pp. 1186-1194, and to the review by Charles L. Hatheway in Chapter 1 of the book entitled Botulinum Neurotoxin and Tetanus Toxin, L. L. Simpson, Ed., published by Academic Press Inc. of San Diego, Calif., 1989, the disclosures in which are incorporated herein by reference.
The neurotoxic component of C. botulinum has a molecular weight of about 150 kilodaltons (kD) and is comprised of a polypeptide chain of about 50 kD (Lc) which is considered to be responsible for the toxic properties of the toxin (i.e., by interfering with the exocytosis of acetylcholine, by decreasing the frequency of acetylcholine release) and a larger polypeptide chain of about 100 kD (Hc), which is believed to be necessary to enable the toxin to bind to the pre-synaptic membrane and to enter into neurons. The neurotoxin gene clusters for C. botulinum serotypes A, B, E and F are believed to be located on the chromosome, while the neurotoxin gene clusters for C. botulinum serotypes C1 and D are carried on bacteriophages [4]. In C. botulinum BoNT/G, the neurotoxin gene (cntA/G) was shown to reside on a large plasmid of approximately 114 kb [5].
Four BoNT/A subtypes (A1-A4) have recently been identified [6, 7, 8]. Although BoNT/A subtypes exhibit a high degree of sequence conservation, variations in substrate recognition and receptor binding regions are sufficient to significantly affect efficient vaccine and countermeasure development [6, 8], as well as potentially affecting pharmaceutical activity. It has also been observed that different type A strains, even within the same subtype, produce varying quantities of BoNT [9]. For instance, experiments have determined that C. botulinum subtype A3 strain Loch Maree, and subtype A4 strain 657Ba, produce considerably less BoNT than most A1 and A2 subtype strains. Interestingly, the dual neurotoxin strain 657Ba produces primarily BoNT/B and even lower quantities of BoNT/A than the Loch Maree strain. This complicates purification of sufficient quantities of these proteins, as required for fundamental molecular biology and structural studies, as well as commercial and therapeutic uses of the proteins.
Therapeutic uses of BoNT include dystonias, chronic facial pain, strabismus, chronic headache, spastic muscle disorders, and other chronic ailments that require repeated administration of BoNT-complexes. Commercially available pharmaceuticals comprising BoNT-complexes (as distinct from purified neurotoxin), i.e., BOTOX (Allergan, Inc.) are serotype A1. Stereotype B has also been commercially marketed as NEUROBLOC or MYOBLOC (Solstice, Inc.), but is mainly used secondary to stereotype A because of the large number of Units required for treatment and its relatively short duration of action compared to serotype A, which has the longest duration of action compared to the other six serotypes of BoNT. Although it is the predominant currently approved type of BoNT, serotype A is immunogenic, meaning that people become resistant to it after repeated use. Accordingly, a need exists for a BoNT/A subtype that is not neutralized from antibodies present against BoNT/A1 after repeated use, an isolated and purified source thereof, as well as methods of obtaining and using said BoNT/A subtype.